Subcutaneous Administration of Anti-Hepatitis B Antibodies

ABSTRACT

The present application relates to an immunoglobulin preparation comprising anti-hepatitis B antibodies for subcutaneous administration and to uses thereof, including methods for treating, preventing and/or reducing a disease, disorder or condition associated with hepatitis B virus.

The present application claims the benefit of U.S. provisionalapplication No. 61/119,827, filed Dec. 4, 2008, the entire contents ofwhich are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to an immunoglobulin preparationcomprising anti-hepatitis B antibodies for subcutaneous administrationand to uses thereof.

BACKGROUND OF THE INVENTION

Hepatitis B is a potentially life-threatening liver infection caused bythe Hepatitis B virus (HBV). HBV infection can result in acute hepatitis(clinically apparent) or in a chronic disease. The clinicalmanifestations of acute infection include yellowing of the skin and eyes(jaundice); extreme fatigue; dark urine; nausea; diarrhea and vomiting;loss of appetite; and muscles, joints or abdominal pain. About 1-2% ofindividuals suffering from acute hepatitis B die from fulminant hepaticfailure. Chronic HBV infection can stay undetected in the body fordecades before it leads to an end-stage liver disease, includingcirrhosis (scarring of the liver) and hepatocellular carcinoma.

HBV infection has a worldwide distribution. It is estimated that morethan 2 billion of the global population has been infected with HepatitisB virus (HBV). Of these, approximately 350 million have chronic liverinfection and are at risk of serious illness and death from cirrhosisand hepatocellular carcinoma [World Health Organization; Fact sheet No.204; August 2008].

Humans are the only reservoir of HBV. The virus is extremely contagiousand is transmitted by percutaneous and mucosal exposure to blood orother body fluids of an infected person. Common modes of transmissionmay include mother-to-infant (perinatal), use of contaminated needles,contaminated blood transfusions and via unprotected sexual contact withan infected person. In addition, HBV is the major infectiousoccupational hazard of health workers, and most health care workersreceive hepatitis B vaccine.

Advanced cases of viral caused hepatitis result in liver failure andorthotopic liver transplantation (OLT) is the only curative treatment.OLT refers to a procedure in which an impaired liver is removed from thepatient's body and a healthy donor's liver is transplanted instead.However, although the diseased liver is removed, circulating virus mayremain in the serum of the patient and in other body compartments;invade and infect the grafted liver; and consequently cause hepatitisrecurrence. Thus, preventing HBV infection of the grafted liver isessential to avoid recurrence of hepatitis and crucial for maintainingliver function in OLT patients.

Passive immunization with Hepatitis B immunoglobulin (HBIG) has beenshown to markedly reduce the incidence of Hepatitis B recurrence afterliver transplantation and is now a well established approach used in OLTpatients (Filipponi et al “Efficacy, safety, and pharmacokinetics ofintramuscular hepatitis B immune globulin, Igantibe®, for theprophylaxis of viral B hepatitis after liver transplantation”. Dig LiverDis. 2009). HBIG is derived from blood plasma of human donors expressinghigh titers of antibodies specific against hepatitis B surface antigen(anti-HBsAg). The antibodies bind to the surface antigen of the virus;inactivate circulating viruses; and prevent subsequent infection.

Immunoglobulin can be administrated in one of the following routes:intravenously (IV), intramuscularly (IM) or subcutaneously (SC).

Moore and Quinn (“Subcutaneous immunoglobulin replacement therapy forprimary antibody deficiency: advancements into the 21st century”. AnnAllergy Asthma Immunol. 2008; 101:114-121) provide a review onsubcutaneous immunoglobulin therapy for patients with primary antibodydeficiency (PAD). In their report it is indicated that immunoglobulininjected into the subcutaneous tissue has reduced intravenousbioavailability secondary to IgG catabolism and incomplete absorptionfrom the subcutaneous space into the intravascular space. On the onehand according to US recommendations when transitioning from intravenousimmunoglobulin (IVIG) to subcutaneous immunoglobulin (SCIG) therapy inpatients with PAD the monthly IVIG dose is increased by 37% and divideby 4 to determine the weekly SCIG dose. The product is infused inmultiple sites simultaneously with a total volume of up to 25 ml perinfusion site. On the other hand dosing recommendations in Europeancountries consists on a 1:1 dose conversion between WIG and SCIGinfusions. It was also reported by Ochs et al (“Safety and efficacy ofself-administered subcutaneous immunoglobulin in patients with primaryimmunodeficiency diseases”. J Clin Immunol. 2006; 26:265-273) that theaverage increase of the SCIG dose to 137% of that given intravenouslydose is unnecessary and does not appear to confer any additionalbenefits in patients with primary immune deficiency diseases (PIDD).

In OLT patients, HBIG is typically administered by intravenous routeconcurrently with the grafting of the transplanted liver (the anhepaticphase), with subsequent daily dosing during each of the first 7 dayspost-transplantation. In the extended post-transplant period HBIG isadministered monthly on an indefinite basis.

Most of the licensed immunoglobulin preparations are for intramuscularuse (Stiehm et al. “Preparation and use of therapeutic antibodiesprimarily of human origin”. Biologicals. 2008; 36:363-374). Thefollowing are some of the commercially available HBIG formulations whichare indicated for intravenous or intramuscular administration:

Omri-Hep-B™ (manufactured by Omrix Biopharmaceuticals) is prepared fromhuman plasma collected from healthy anti-HBsAg high titer donors.Omri-Hep-B™ is used for passive immunization in the prevention ofhepatitis B recurrence after liver transplantation. Omri-Hep-B™ is asterile solution containing 5% protein of which at least 95% is humanimmunoglobulin G, 50 IU/ml of antibodies to HBsAg as the activeingredient, 10% maltose and water for injection. Omri-Hep-B™ isadministered by intravenous infusion. HepaGam B™ (manufactured byCangene Corporation) is indicated for the prevention of hepatitis Brecurrence following liver transplantation, in HBsAg positive livertransplant patients. It is prepared from selected plasma donated byhealthy, vaccinated and anti-HBsAg high titer donors. HepaGam B™ isformulated as a 5% (50 mg/mL) protein solution with 10% maltose and0.03% polysorbate 80 at pH 5.6. It is available in 1 ml and 5 ml singledose vials containing about 312 IU/ml anti-HBsAg. HepaGam B™ isadministered intravenously.

Hepatec® (manufactured by Biotest Pharma GmbH) is a human hepatitis Bimmunoglobulin solution for intravenous administration. 1 ml Hepatec®contains: 100 mg human plasma protein (10% protein) of which at least95% is immunoglobulin G, 50 IU of antibodies to HBsAg, sodium chlorideand water for injection.

HyperHEP B™ (manufactured by Talecris Biotherapeutics Inc.) is asolution which contains high quantities of anti-hepatitis B immuneglobulin for intramuscular administration. HyperHEP B™ is prepared fromthe plasma of healthy vaccinated anti-HBsAg high titer donors. It isformulated as a 15-18% protein solution at a pH of 6.4-7.2 in 0.21-0.32M glycine. The formulation contains about 220 IU per ml anti-HBsAg.BayHep B® (distributed by Bayer New Zealand Limited) is a hepatitis Bimmunoglobulin solution for intramuscular injection. 1 ml contains 217IU anti-hepatitis B antibodies and 19.5 mg glycine. It is available in0.5 ml neonatal single dose syringe, and 1 ml single dose vial.

Nabi-HB® (manufactured by Nabi® Biopharmaceuticals) is a sterilesolution of immunoglobulin (5±1% protein) containing antibodies tohepatitis B surface antigen (anti-HBs). It is prepared from plasmadonated by individuals with high titers of anti-HBs. Nabi-HB isformulated in 0.075 M sodium chloride, 0.15 M glycine, and 0.01%polysorbate 80, at pH 6.2. It contains 312 IU/ml anti-HBs and it isintended for administration in the intramuscular route.

According to the leaflet HyperHEP B™, BayHep B® and Nabi-HB® areindicated for acute exposure to blood containing HBsAg; perinatalexposure of infants born to HBsAg-positive mothers; sexual exposure toan HBsAg-positive person; and household exposure to persons with acuteHBV infection.

Igantibe® (manufactured by Grifols) is a human anti-hepatitis Bimmunoglobulin for intramuscular use. Igantibe® is obtained fromimmunized plasma donors. The product contains 200 IU/ml with an IgGcontent of higher than 95% and HBIG content of 15.3%. It is formulatedwith glycine (2.25%), sodium chloride (0.30%) and sterile water forinjection.

Various IV and/or IM dosing regimes of HBIG optionally combined with ananti-viral drug such as lamivudine are reported for OLT subjects.

Filipponi et al (2009) reported administration of Igantibe®, a specificHBIG (manufactured by Grifols), to adult patients who underwent livertransplantation more than 18 months earlier. IM administration of 2000IU Igantibe® was carried out once every 14 days namely bimonthly for aperiod of 6 months. This regime allowed anti-HBs levels of ≧150 IU/L inthe circulation of the subjects throughout the study period. In anotherstudy, Igantibe® was administered IM to OLT patients in a one monthlydose of 2000 IU and the HBV antibody titer were measured every month.This regime attained a protective antibody titer of >100 IU/L in thecirculation for at least the first year after transplantation (Alonso etal. “Effectiveness of low-dose intramuscular anti-VHB immune globulin inthe prophylaxis of viral B hepatitis reinfection after livertransplantation: preliminary report”.

Transplant Proc. 2003; 35:1850-1851). Both regimes (i.e. bimonthly andmonthly administration) were considered protective and efficacious inprophylaxis of viral B hepatitis after liver transplantation.

Also, Faust et al. (“Cost-effective and safe ambulatory long-termimmunoprophylaxis with intramuscular instead of intravenous hepatitis Bimmunoglobulin to prevent reinfection after orthotopic livertransplantation”. Clin Transplant. 2003; 17:254-258) reported thatadministration of 2000 IU IM HBIG given once a month together with 100mg lamivudine per day is effective in prophylaxis of HBV re-infection inpatients after OLT.

Yao et al. (“Intramuscular hepatitis B immune globulin combined withlamivudine for prophylaxis against hepatitis B recurrence after livertransplantation”. Liver Transpl Surg. 1999; 5:491-496) described aprotocol comprising administration of IM HBIG and oral lamivudine (150mg/d) in patients with HBV infection who underwent OLT. Patients withdetectable HBV DNA at the time of OLT received 10,000 U (45 ml) of IVHBIG daily for 7 days, followed by 5 ml of IM HBIG weekly for the next 3weeks, then every 3 weeks. Patients who were HBV DNA negative receivedon dose of IV HBIG (45 ml) during surgery, followed by 5 ml IM HBIGweekly for 4 weeks, then every 3 weeks. During a follow up of 15.6months, all patients developed protective anti-HBs titers greater than200 IU/L in the circulation and had no evidence of HBV recurrence.

Yoshida et al (“Liver transplantation for chronic hepatitis B infectionwith the use of combination lamivudine and low-dose hepatitis B immuneglobulin”. Liver Transpl Surg. 1999; 5:520-5.) studied the effectivenessof a post-transplantation prophylaxis protocol using a combination oflamivudine and HBIG in patients with chronic HBV infection. In themaintenance period HBIG therapy consisted of 2170 IU intramuscularlytwice weekly, tapered to every 2 to 4 weeks by 12 monthspost-transplantation. Target serum HBIG titers were less than 500 IU/Lfor the first 6 months after OLT and then greater than 300 IU/L withinthe following 6 months. All patients achieved the target HBIG titer andallograft re-infection by HBV was prevented.

Using subcutaneous administration of immunoglobulin has severaladvantages. For example, subcutaneous IG infusions are well toleratedcompared to IM administration, safe, and give the patient moreindependence compared to IV administration.

The reported dose for subcutaneous HBIG administration is similar orlower than that of intramuscular administration.

Subcutaneous administration of hepatitis B immunoglobulin (HBIG) incombination with lamivudine was previously reported (Powell et al.(“Subcutaneous administration of hepatitis B immune globulin incombination with lamivudine following orthotopic liver transplantation:effective prophylaxis against recurrence”. Clin Transplant. 2006;20:524-525). According to the report the disclosed HBIG contained anantibody titer of 217 IU/ml anti-HBsAg and a regime of multiple dailyinjections (four aliquots of 2.5 ml) was applied via the subcutaneousroute for seven days following the liver transplantation. Maintenancesubcutaneous dosing of 2170 IU was administered every three weeksachieving serum anti-HBs titers from 500 to 1000 IU/L over five monthspost transplant.

The following reports show pharmacokinetic parameters of subcutaneousHBIG vis a vis intramuscular administration.

Thürmann et al. (“Pharmacokinetics and safety of a novelanti-HBs-enriched immunoglobulin in healthy volunteers aftersubcutaneous and intramuscular administration”. Eur J Clin Pharmacol.2006; 62:511-512) investigated the pharmacokinetics of ananti-HBsAg-enriched immunoglobulin preparation which contains 635 IUanti-HBsAg/ml. The immunoglobulin was administered in an equal singledose of 30 IU/kg body weight either subcutaneously or intramuscularly.According to the paper comparable serum concentrations were obtainedusing both subcutaneous and intramuscular administration routes.Additionally, the bioavailability of the investigated HBIG preparationfollowing subcutaneous and intramuscular administration was comparableto that following intravenous administration.

In a recent study Hooman et al. (“Antibody to hepatitis B surfaceantigen trough levels and half-lives do not differ after intravenous andintramuscular hepatitis B immunoglobulin administration after livertransplantation”. Liver Transpl. 2008; 14:435-442) comparedpharmacokinetic parameters after IM and IV administration of 2000 IUHBIG. According to the report, although the area under the curve (AUC)levels were not measured during the first two weeks after HBIGadministration, it was presumed that they were lower in IM as comparedto IV administration. It is also indicated that the principles ofpharmacokinetic parameters after SC administration are expected to besimilar to IM administration. The report is silent on the levels ofanti-HBs present in the circulation during the first two weeks followingthe different administration routes.

Recently (September 2009), the European Committee for Medical Productsfor Human Use (CHMP) recommended the granting of a marketingauthorization for Zutectra®, a human hepatitis B immunoglobulin solutionfor subcutaneous administration. Zutectra® is manufactured from plasmaof donors with high anti-HBs antibody titers (Biotest Pharma GmbH) andis intended for the prevention of hepatitis B virus re-infection afterliver transplantation. It is available in 1 ml pre-filled syringecontaining 500 IU anti-HBs antibodies. It is reported that effectiveanti-HBs-serum levels are achieved with weekly Zutectra® application.

SUMMARY OF THE INVENTION

Administration of hepatitis B immunoglobulin (HBIG) reduces thefrequency and severity of hepatitis B virus (HBV) infection. In thisregard, maintaining adequate levels of anti-hepatitis B antibodies inthe circulation of a subject in need is highly desired.

Advanced cases of viral caused hepatitis result in liver failure andorthotopic liver transplantation (OLT) is the only curative treatment.OLT refers to a procedure in which an impaired liver is removed from thepatient's body and a healthy donor's liver is transplanted instead.However, although the diseased liver is removed, circulating virus mayremain in the serum of the patient and in other body compartments;invade and infect the grafted liver; and consequently cause hepatitisrecurrence. Thus, preventing HBV infection of the grafted liver isessential to avoid recurrence of hepatitis and crucial for maintainingliver function in OLT patients.

Passive immunization with intravenous Hepatitis B immunoglobulin (HBIG)has been shown to markedly reduce the incidence of Hepatitis Brecurrence after liver transplantation and is now a well establishedapproach used in OLT patients.

To prevent or minimize HBV recurrence in patients who have undergoneorthotopic liver transplantation, HBIG therapy is required on a lifelongbasis. Oftentimes, HBIG is administered via the intravenous orintramuscular route. In some cases the intravenous formulation is usedintramuscularly.

Using subcutaneous therapy for these patients offers several advantagesover the two other routes of administration.

An intravenous therapy requires administration by a trained medicalpersonal and at times hospitalization. In contrast, in subcutaneoustherapy close supervision is unnecessary, and infection is unlikely tooccur. Subcutaneous therapy can be self administered at home,significantly improving the patient's quality of life.

Furthermore, following organ transplantations patients are regularlyunder immunosuppressive regimes (Ringe et al. “A novel managementstrategy of steroid-free immunosuppression after liver transplantation:efficacy and safety of tacrolimus and mycophenolate mofetil”.Transplantation. 2001; 71:508-515). These immunocompromised patients areat high risk of contracting an infectious disease. Thus, avoidingprolonged intravenous drug administration and hospital admission is ofgreat advantage.

Although intramuscular injection, as subcutaneous administration, can beself-administered, it causes significant pain at the injection site, aphenomenon which leads to poor compliance in some patients (Moore andQuinn 2008). Also, intramuscular injection may cause deep tissuecomplications.

The art discloses administering HBIG preparations by subcutaneous andintramuscular routes. The art does not differentiate between these tworoutes of administration and in some cases the intramuscularformulation/dose is used subcutaneously.

It was found according to the present invention that subcutaneousinjection results in a delayed appearance of HBIG in the circulation ascompared to intramuscular administration. These findings pave the way toa different treatment approach when using HBIG by the subcutaneousroute. The present invention provides administration of a HBIGsubcutaneous dose which is higher than the dose that is required oremployed by the intramuscular route, e.g. 1.5, 2 or 2.5 folds higher.

Advantageously, the present invention provides an improved hepatitis Bimmunoglobulin preparation for subcutaneous administration and methodsfor subcutaneous administration of hepatitis B immunoglobulin.

In one aspect, the invention provides a method for subcutaneousimmunoglobulin hepatitis B therapy, comprising subcutaneouslyadministering to a subject in need a dose of anti-hepatitis Bimmunoglobulin that is higher than a dose of anti-hepatitis Bimmunoglobulin administered by the intramuscular route, therebyachieving anti-hepatitis B antibodies in the circulation at a similarlevel and timing as by the intramuscular route.

In one embodiment of the invention, the subject is selected from thegroup consisting of an orthotopic liver transplantation (OLT) patient, anewborn to a viral infected mother, and a subject exposed to the virus.

In another embodiment of the invention, the subcutaneous dose is atleast 2-fold higher than the dose administered by the intramuscularroute.

Yet, in another embodiment of the invention, the subcutaneous dose ishigher than 2170 IU.

In a further embodiment of the invention, the subcutaneous dose is equalto or higher than 3000 IU.

In another embodiment of the invention, the subcutaneous administeringis carried out by injection.

In another further embodiment of the invention, the subcutaneousadministering is carried out by a single dispensation.

Yet, in another embodiment of the invention, the subcutaneous dose has atiter of anti-hepatitis B antibodies that is greater than 635 IU/ml.

Another aspect of the invention relates to a method for treating,preventing and/or reducing a disease, disorder or condition associatedwith hepatitis B virus, comprising administering to a subject in needthereof a dose of anti-hepatitis B immunoglobulin sufficient to obtain apredetermined level of anti-hepatitis B antibodies in the circulation ofthe subject, the improvement wherein the anti-hepatitis B immunoglobulinis administered by the subcutaneous route in a dose that is at least 1.5times higher than the intramuscular formulated dose needed to obtainsaid predetermined level of anti-hepatitis B antibodies in thecirculation of the subject, wherein the predetermined level is obtainedin the circulation by the subcutaneous dose at a similar timing as inthe intramuscular formulated dose.

In one embodiment of the invention, the predetermined level ofanti-hepatitis B antibodies in the circulation is greater than 100mIU/ml.

In another embodiment of the invention, the timing is before the third,second or first day following subcutaneous administration.

In still another embodiment of the invention, the subcutaneousadministration is carried out by injection.

In yet another embodiment of the invention, the subcutaneousadministration is carried out by a single dispensation.

In yet another embodiment of the invention, the subject was previouslytreated by a dosing regimen of intravenous administration ofanti-hepatitis B immunoglobulin.

In another embodiment of the invention, the subcutaneous dose has atiter of anti-hepatitis B antibodies that is greater than 635 IU/ml.

Another object of the invention is to provide a method for preventinghepatitis B viral infection recurrence in a subject who underwentorthotopic liver transplantation (OLT), comprising administering to thesubject a dose of anti-hepatitis B immunoglobulin sufficient to obtain apredetermined level of anti-hepatitis B antibodies in the circulation ofthe subject, the improvement wherein the anti-hepatitis B immunoglobulinis administered by the subcutaneous route in a dose that is at least 1.5times higher than the intramuscular formulated dose needed to obtain asimilar level of anti-hepatitis B antibodies in the circulation of thesubject, wherein the predetermined level is obtained in the circulationby the subcutaneous dose at a similar timing as in the intramuscularformulated dose.

In one embodiment of the invention, the subcutaneous administering iscarried out by injection.

In another embodiment of the invention, the subcutaneous administeringis carried out by a single dispensation.

A further aspect of the invention is to provide a method for treating,preventing and/or reducing a disease, disorder or condition associatedwith hepatitis B virus in a subject in need thereof, the methodcomprising a first dosing regimen of intramuscular administration ofanti-hepatitis B immunoglobulin, followed by a second dosing regimen,which comprises subcutaneously administering to the subject a dose ofanti-hepatitis B immunoglobulin that is higher than the dose employed bythe first dosing regimen of intramuscular administration, therebyobtaining anti-hepatitis B antibodies in the circulation of the subjectat a similar level and timing as in the first dosing regimen ofintramuscular administration.

In one embodiment of the invention, the timing is before the third,second or first day following subcutaneous administration.

BRIEF DESCRIPTION OF THE DRAWINGS

The features, aspects, and advantages of the present invention willbecome better understood with regard to the following description,examples, claims, and the following figures.

FIG. 1: shows the recovery of anti-hepatitis B antibodies in rabbit'sblood following administration of Hepatitis B immunoglobulin (HBIG). Theimmunoglobulin was administered by intravenous (IV), intramuscular (IM)or subcutaneous (SC) route. The level of antibodies on day one followingIV administration was assigned as a reference value (100%).

FIG. 2: shows the level of the anti-hepatitis B antibodies in rabbit'sblood during the first two weeks following administration of HBIG. Theimmunoglobulin was administered by IV, IM or SC route.

FIG. 3: shows the recovery of anti-hepatitis B antibodies in rat's bloodfollowing administration of HBIG. The immunoglobulin was administered byIV, IM or SC route. The results are presented as percentage of the levelof antibodies on day one following IV administration (100%).

FIG. 4: shows the anti-hepatitis B antibody levels in rat's blood during15 days following administration of HBIG. The immunoglobulin wasadministered by IV, IM or SC route.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for subcutaneous administrationof hepatitis B immunoglobulin and an improved hepatitis B immunoglobulinpreparation for subcutaneous administration. More specifically, thepresent invention relates to the use of an increased subcutaneous doseof anti-hepatitis B immunoglobulin when transitioning from intramuscularto subcutaneous therapy.

In one aspect, the invention provides a method for achieving anefficient level of anti-hepatitis B antibodies in the circulation of asubject in need through subcutaneous administration. In one embodimentof the invention, the method comprises a) providing an anti-hepatitis Bimmunoglobulin preparation, b) administering subcutaneously to saidsubject the preparation of a), wherein said preparation is administeredat a dose that is higher than that applied by intramuscular route.

The term “subcutaneous administration” refers to introduction e.g. byinjection of the immunoglobulin preparation under the skin of an animalor human patient. The introduction of the preparation can be carried outby creating a pocket between the skin and the underlying tissue. Theadministration can be to the layer of fat which lies between the dermisand underlying fascia. The pocket may be created by pinching or drawingthe skin up and away from underlying tissue. Places on the body wherepeople can administer the immunoglobulin include, but are not limitedto, the outer area of the upper arm, above or below the waist, the upperarea of the buttock, just behind the hip bone and the front of thethigh. In one embodiment of the invention, the subcutaneousadministration is carried out by injection.

Within the context of this invention the term “dose” refers to theamount of antibody given in a single administration. The term“administration”, as used herein, means introduction of theimmunoglobulin composition into the subject by subcutaneousadministration.

The invention teaches that when transitioning from intramuscular tosubcutaneous therapy with hepatitis B antibody preparation the doseneeds to be adjusted accordingly. In one embodiment of the invention,the required subcutaneous dose is at least 1.5-fold, such as about 2 or2.5 fold the intramuscular dose.

The teaching of the invention is based on results obtained according tothe invention. It was surprisingly found according to the presentinvention that administering anti-HBsAg by the subcutaneous routeresults in a decreased bioavailability on the first two days followingadministration as compared to the intramuscular route. Also, it has beenfound according to the present invention that in the subcutaneous routethe antibodies are slowly released into the intravascular spaceachieving a peak serum concentration 3 to 4 days after administration.More specifically, the results according to the invention show thatintramuscular administration achieved efficient levels faster thansubcutaneous administration. The term “bioavailability” refers to thefraction of an administered dose that reaches the general circulation.

These findings point toward the advantage of administeringsubcutaneously a higher amount of antibodies as compared to theintramuscular route to rapidly achieve efficient levels of antibodies inthe circulation of the subject.

There are patients who after administration of an immunoglobulinpreparation fail to achieve efficient levels of anti-HB antibodies inthe circulation and patients who are particularly susceptible toextensive loss of circulated anti-hepatitis B antibodies, for example,patients who have surgical bleeding, patients having undergone abdominalfluid drainage, and patients who undergo plasmapheresis. Thepreparations and methods according to the invention are of cardinalimportance in these patients. These patients may require a higher doseand/or more frequent injections. For example, the subcutaneous dose inthese patients can be at least 2-fold, such about 2.5 or 3 fold theintramuscular dose. Also, the preparations and methods of the inventioncan be advantageously used in patients with poor intravenous access,patients with aseptic meningitis, patients with anaphylactic reactionsto intravenous immunoglobulin, patients with rapid gastrointestinalprotein loss, and immunocompromised patients such as patients followingorgan transplantations receiving immunosuppressive regimes and patientsreceiving anti-cancer treatment.

In one embodiment of the invention, the titer of the anti-hepatitis Bantibodies in the immunoglobulin preparation is higher than 635 IU/ml,for example, equal to or greater than 700, 1000, 1400 or 2000 IU/ml.

Subcutaneous IgG are usually administered by continuous slow infusionusing an infusion portable pump. In addition, administration of largedoses is accomplished by using multiple application sites (Stiehm et al.2008). One of the advantages of the immunoglobulin preparation of theinvention is that it contains a high concentration of anti-hepatitis Bantibodies which enables subcutaneous injection of the required highdose in a low volume without the need of a continuous slow infusionand/or multiple applications such as in a single dispensation. Forexample, the dose for an adult weighing 70 kg can be administered at avolume of equal to or less than about 4.5 ml, for example, about 3, 2,1.5 or 1 ml.

As used herein, the term “infusion” refers to a continuous, slowintroduction of fluids into the body. The term “infusion portable pump”refers to low infusion rate pressurizing devices used in administeringor infusing medical fluids.

In the results according to the invention, when comparing theanti-hepatitis B antibodies in the circulation on day one followingadministration, it is evident that IM administration lead to about2-fold increase in the antibody levels as compared to SC administration.The results obtained herein collected during the observation period oftwo weeks show that the calculated average area under the curve (AUC) inboth models was similar in the SC and IM routes. Administeringsubcutaneously a higher amount of antibodies may increase the AUC.Employing higher amounts of antibodies can be advantageously used toobtain early adequate serum IgG levels.

Using the method according to the invention will assist to quicklyobtain an adequate level of immunoglobulin in the circulation of apatient. Also, subcutaneous administration according to the inventionwill result in anti-hepatitis B antibodies in the circulation at asimilar level and timing as the intramuscular administration. In oneembodiment of the invention, the similar level is achieved before thethird day following subcutaneous administration of the immunoglobulinpreparation. In another embodiment of the invention, said level isreached on the second day post subcutaneous administration. Yet, inanother embodiment of the invention, said level is reached one dayfollowing subcutaneous administration of the immunoglobulin preparation.

As used herein, the terms “efficient level” and “adequate level” referto an amount of anti-hepatitis B antibodies in the circulation of ananimal or human patient that is similar to the level of anti-hepatitis Bantibodies following IM administration and/or that will elicit a“therapeutic response”. As used herein the term “therapeutic response”means alleviating the symptoms of the HBV infection, inhibitinghepatitis recurrence in OLT patients and/or reducing the number oreliminating the hepatitis B viral particles in the circulation of thepatient.

Alonso et al. (2003) reported that an antibody titer of >100 mIU/ml inthe circulation is considered protective and efficacious in prophylaxisof viral B hepatitis. Accordingly, the efficient level of anti-hepatitisB antibodies in the circulation can be about or greater than 100 mIU/ml,like for example 150, 200, 300, 400 or 500 mIU/ml. In one embodiment ofthe invention, the level is about or greater than 100 mIU/ml. In anotherembodiment of the invention, the efficient level is higher than 100mIU/ml. Obtaining quickly levels of anti-hepatitis B antibodies higherthan 100 mIU/ml such as 150, 200, 250, 300 and 500 mIU/ml is ofadvantage in high risk OLT patients and/or in patients in whichadministration of antiviral agents is not possible. The term “high riskOLT patients” is for example patients with detectable HBV E antigen(HBeAg) prior to liver transplantation and/or patients who areparticularly susceptible to extensive loss of circulating anti-HBs suchas patients who have surgical bleeding, patients who undergo abdominalfluid drainage, and patients who undergo plasmapheresis.

The term “hepatitis B viral infection” refers to “acute hepatitis B” or“chronic HBV” infection which may develop to permanent liver damage(scarring of the liver), end-stage liver disease, and/or hepatocellularcarcinoma. By “acute hepatitis B” it is meant a new symptomatic HBVinfection. Symptoms and signs of the acute disease include, but are notlimited to, loss of appetite, nausea, weakness, abdominal and jointpain, vomiting, jaundice (yellow eyes and skin), skin rashes and darkurine. By “chronic HBV infection” it is meant persistent i.e. long terminfection with HBV. The chronic infection may be present withoutapparent symptoms.

According to the invention, the monthly dosage of anti-HBsAg can be morethan 30 IU/kg such as 42, 70, 85, 94 IU/kg body weight. In oneembodiment of the invention, the monthly dosage is about 42 IU/kg bodyweight. In another embodiment of the invention, the dosage per month foran adult weighing 70 kg is more than 2100 IU such as 2170 IU. Yet inanother embodiment of the invention, the dosage per month for a childweighing 20 kg is more than 600 IU. Yet in another embodiment of theinvention, the dosage per month for an adult weighing 70 kg is about3000 IU. Yet in another further embodiment of the invention, the dosageper month for an adult weighing 70 kg is about 4950 IU. In anotherembodiment of the invention, the dosage per month for an adult weighing70 kg is about 6000 IU.

The term “dosage” and “dosing” refers to a schedule regimen wherein oneor multiple doses of the compositions are administered during apredetermined period of time. In one embodiment of the invention theperiod of time is one month.

The total dosage per month can be administered in one injection or canbe divided to several portions and administered several times during themonth while at least the initial portion is administered at a higherdose than that required by intramuscular route, such as 1.5, 2 or 2.5folds higher. For example, the total monthly treatment can be divided totwo and administered once every two weeks. In one embodiment of theinvention, each subcutaneous dose is administered at a higher dose thanthat required by or employed with intramuscular route. In anotherembodiment of the invention, each subcutaneous dose is administered at adose which is at least 1.5-folds higher than that required by oremployed with intramuscular route.

In one embodiment of the invention, the immunoglobulin is administeredpre or post orthotopic liver transplantation. In another embodiment ofthe invention, the immunoglobulin is administered for prophylaxis ofhepatitis B viral infection post orthotopic liver transplantation. Inthe latter embodiment the immunoglobulin can be administered during themaintenance treatment period. The term “maintenance period” refers tothe post-transplant time point wherein a single dose is administered atrepeated intervals (referred to herein as maintenance dose) to maintaina protective level of anti-hepatitis B in the circulation and decreasethe risk of viral hepatitis B recurrence. The maintenance period canrefer to the early post-transplant time point such as the first sevendays following the liver transplantation or the extended post-transplantperiod such as eight days onwards. In one embodiment of the invention,the maintenance period refers to the extended post-transplant period.

Advantageously, the IM dosing and the maintenance period have been shownin the literature and the art to achieve protective levels ofanti-hepatitis B in the circulation and/or to statistically preventhepatitis B virus re-infection in subjects who underwent orthotopicliver transplantation. According to the invention when transitioningfrom intramuscular to subcutaneous administration the administered doseshould be increased, for example, the dose is at least 150% of thatgiven intramuscularly. The recommended IM dose can change for a givenindication, and for a desired level in the circulation (e.g. 100 vs. 500mIU/ml). The subcutaneous dose should be increased to achieve the samepharmacokinetic characteristics as each recommended IM dose.

For example, to achieve an anti-HBs level of greater than 100 mIU/ml theimmunoglobulin preparation can be administered subcutaneously once amonth at a dose of 3000 IU. Alternatively, to achieve an anti-HBs levelgreater than 150 mIU/ml in the circulation, the immunoglobulinpreparation can be administered subcutaneously bi-monthly each at a doseof 3000 IU. In another embodiment of the invention, to obtain anti-HBstiters greater than 200 mIU/ml in viremic patients (patients who are HBVDNA positive), the immunoglobulin preparation can be subcutaneouslyadministered at a weekly dose of 1650 IU on week 2-4 post-operative andthen at a dose of 1650 IU every three weeks. In another furtherembodiment of the invention, to obtain anti-HBs titers greater than 200mIU/ml in HBV DNA negative patients the immunoglobulin can beadministered at a dose of 1650 IU on week 1 to 4 post-operative and thenevery 3 weeks. In another further embodiment of the invention, theimmunoglobulin preparation can be administered at a dose of 3255 IUtwice weekly, followed by a dose of 3255 IU every 2 to 4 weeks by 12months post-transplantation. Advantageously, this treatment may achievea target serum HBIG titer of equal to or less than 500 mIU/ml in thefirst 6 months after OLT and then greater than 300 mIU/ml within thefollowing 6 months.

The administered immunoglobulin preparation may optionally comprise anacceptable carrier. As used herein the term “acceptable carrier” refersto a diluent or a vehicle which is suitable for subcutaneous injection.The carrier can be selected from any of the carriers known in the artsuch as, but not limited to, saline, water, emulsions, sodium chloridesolution, and mixtures of organic solvents.

The immunoglobulin preparation can also comprise excipients. As usedherein the terms “excipient” refers to an inert substance which is addedto the pharmaceutical composition. The excipients can be added into thepreparation, for example, in order to ensure that the active ingredientretains its chemical stability and biological activity upon storage, toaid the manufacturing process and/or for aesthetic reasons e.g. color.

Examples of excipients include, but are not limited to, various sugars,such as maltose, sucrose or, D-sorbitol; glycine; polymeric excipients,such as PEG or serum proteins, such as albumin.

In one embodiment of the invention, the immunoglobulin preparationcomprises from about 1.6 to about 2.6% glycine, such as about 2.25%, and0.3% NaCl.

The immunoglobulin preparation can comprise a relatively high proteinconcentration. The range of protein concentration can be from about 5 to25% w/v, from about 15 to 25% w/v, from about 14 to 20% w/v, or fromabout 14 to 18% w/v. In one embodiment of the invention, the preparationof the invention has a protein concentration of about 16% w/v.Advantageously, the preparation is well tolerated when administeredsubcutaneously. In one embodiment of the invention, at least 90%, forexample about 91, 92, 93, 94, 95, 96, 97, 98 or 99% of the proteins inthe preparation are human immunoglobulin G. The immunoglobulinpreparation of the invention can contain equal to or less than 10%polymers and/or aggregates, for example, the polymer levels can be lessthan 3%. The content of monomers and/or dimers in the preparation can beequal or more than 85%. Optionally, the immunoglobulin preparation isadjusted to a physiological compatible value. The pH range can bebetween about 6.4 to about 7.2, for example about 6.4, 6.5, 6.6, 6.7,6.8, 6.9, 7, 7.1 or 7.2. The pH of the preparation can be near neutral.

The method according to the invention can be used for preventing,treating, and/or reducing hepatitis B viral infection.

The term “treating” refers to curing, healing, alleviating, relieving,altering, remedying, substantially ameliorating, improving, or affectinga disease, disorder or condition associated with hepatitis B virus, orone or more symptoms thereof. As used herein, “preventing”, and“prophylaxis” refers to reducing the risk or likelihood that the subjectwill acquire a disease, disorder or condition associated with hepatitisB virus. The term “reducing” refers to decreasing the progression orseverity of the disease.

In one embodiment of the invention, the method is used to enhance thebioavailability of hepatitis B immunoglobulin when administeredsubcutaneously. In another embodiment of the invention, the method isused to prevent viral hepatitis B recurrence following orthotopic livertransplantation (OLT). In another embodiment of the invention, themethods of the invention are used for preventing viral hepatitis B inorthotopic liver transplantation (OLT) patients. In another furtherembodiment of the invention, the methods of the invention are used in asubject born to a hepatitis B viral infected mother. Yet, in anotherembodiment of the invention, the methods of the invention are used in asubject exposed to hepatitis B virus.

The present invention provides a method for preventing hepatitis B viralinfection recurrence in a subject who underwent orthotopic livertransplantation. The method comprises administering subcutaneously tothe subject an anti-hepatitis B immunoglobulin preparation. In oneembodiment of the invention the administering comprises a maintenancedose which is higher than the intramuscular dose, thereby achievinganti-hepatitis B antibodies in the circulation of the subject at asimilar level and timing as in the intramuscular route.

In one embodiment of the invention, the required maintenancesubcutaneous dose is at least 1.5-fold, such as about 2 or 2.5 fold theapplied maintenance intramuscular dose as exemplified above.

The immunoglobulin preparation can comprise anti-hepatitis B antibodiesat a titer of greater than 635 IU/ml such as about 700, 1000, 1400, and2000 IU/ml.

Subject matter of the present invention embraces an immunoglobulinpreparation for subcutaneous administration as specified above. Theimmunoglobulin preparation contains hepatitis B antibodies which reactwith the surface antigen(s) or determinant(s) of the virus and destroysand/or inhibits its infectivity and virulence. The immunoglobulinpreparation can be used in a subject pre or post OLT due to a liverdisease caused by hepatitis B viral infectiuon.

The immunoglobulin preparation of the invention can be prepared fromblood or blood fractions donated by healthy, screened donors with hightiters of anti-hepatitis B antibodies. Example of such a technique isdisclosed in WO-2007/017859 which content is incorporated herein byreference. The blood or blood fraction can be human or of non-humansource.

The term “blood fraction” refers to a fraction of whole blood whichcomprises immunoglobulins such as plasma or serum.

The cut-off value of the blood or blood fraction can be 40 IU/ml. In oneembodiment of the invention, the cut-off value is 50 IU/ml. In anotherembodiment of the invention, the cut-off value is 80 IU/ml. A “cutoff”value is the minimum value of blood fraction sample which is consideredas a high titer; thus being suitable for manufacturing theimmunoglobulin preparation of the invention.

The presence of anti-hepatitis B antibodies in the blood sample can bemeasured by a variety of known methods, such as biochemical assays orbiological assays.

In one embodiment of the invention a bioassay such as plaque reductionneutralization tests (PRNTs) can be used in order to determine theantibody levels. The term PRNT refers to a reduction in plaque counts,e.g. a PRNT50 titer is based on a 50% or greater reduction in plaquecounts. The assay can be performed in an in-vitro setting in variouscell types. In another embodiment of the invention a biochemical assaysuch as Enzyme-Linked Immunosorbent Assay (ELISA) is carried out inorder to determine the antibody levels. Several variations of ELISAtesting can be used, including, but not limited to, indirect ELISA,sandwich ELISA, and competitive binding. Also, an automated ELISA systemcan be used, for example by using an automated instrument, in order toallow multiple screening.

The plasma pool can be treated to reduce and/or inactivate infectiveparticles. This can be carried out by different methods such as, but nolimited to, filtration, solvent/detergent treatment, heat treatment,such as pasteurization, gamma or UVC (<280 nm) irradiation, or by anyother method known in the art.

The term “infective particle” refers to a microscopic particle, such as,but not limited to, a microorganism or a prion, which can infect orpropagate in cells of a biological organism. The infective particles arefor example viral particles.

The inactivation procedure of infective particles can be carried out byadding an inactivating molecule to the composition prior to and/orduring the procedure. The added molecules and their products can beremoved by gravitation, column chromatography, phase separation or byany other method known in the art. The removal of infective particlescan be carried out by filtration or by selective absorption methods suchas affinity, ion exchange or hydrophobic chromatography. A multi-stepviral inactivation procedure can be carried out. For example, thecomposition can be subjected to solvent/detergent treatment, heattreatment, selective chromatography and filtration. Examples ofimmunoglobulin purification techniques are disclosed in U.S. Pat. No.6,468,733, EP patent No. 1,161,958 and International PCT PublicationWO99/18130 whose contents are incorporated by reference. For example, amethod for the purification of immunoglobulins from a source solutionsuch as Cohn Fraction II may comprise: (a) pre-treating a cationexchange resin with an acidic solution having a pH of 4.0-4.5; (b)contacting the source solution with the cation exchange resin; and (c)eluting the immunoglobulins bound to the cation exchange resin. Prior tocontact with the cation exchange resin, the source solution may betreated with an organic solvent and detergent.

The immunoglobulin preparation can be concentrated by ultra-filtrationprocess. The ultrafiltration can be followed by diafiltration toexchange the buffer. The concentration and dialysis by ultrafiltrationand diafiltration, respectively, can be performed in one step or as twoseparate steps. The diafiltration can be carried out against anysolution which is suitable for subcutaneous injection. Non limitingexamples of such solutions include, but are not limited to, 0.3% NaCland from about 1.6 to about 2.6% glycine such as about 2.25%.

Regular monitoring of serum hepatitis B antigen such as hepatitis Bsurface antigen (HBsAg) and/or levels of anti-hepatitis B antibodyfollowing administration of the immunoglobulin preparation can becarried out to track treatment response and allow for treatmentadjustment. The antibody levels can be determined by a variety ofmethods as indicated above and hepatitis B antigen presence in thecirculation can be determined using techniques well known in the artsuch as an ELISA procedure as indicated above. In one embodiment of theinvention, the response to the therapy is examined by monitoring theserum HBV DNA levels. In another embodiment of the invention, theresponse is examined by monitoring loss of hepatitis e antigen (HBeAg)in patients who were initially HBeAg positive. Alternatively, liverfunction tests can be carried out to assess treatment response such asmeasuring alanine transaminase (ALT) serum levels.

The immunoglobulin preparation according to the invention can beprovided in a vial or pre-filled syringe. The immunoglobulin preparationcan be stored in either a liquid or in a solid form, e.g. as lyophilizedpowder. The preparation can be stored at a temperature of 4° C. Thepreparation of the invention may be used, for example, for passiveimmunization of subjects pre or post liver transplantation such as forprophylaxis of patients who underwent orthotopic liver transplantation,for passive immunization of individuals who are at increased risk ofinfection with hepatitis B virus such as, but not limited to, babiesborn to a hepatitis B viral infected mother, subject exposed to HBV, forexample, by parenteral exposure (e.g., accidental “needle stick” of ahealth care worker), direct mucous membrane contact (e.g., accidentalsplash), or oral ingestion (e.g., pipetting accident) involvinghepatitis B-infected material such as blood, plasma or serum.

The immunoglobulin preparation can also be used as prophylaxis insubjects who are unable to develop adequate immune protection and whoare exposed to continual risk of infection, e.g. dialysis patients,patients receiving multiple transfusion or blood components.

Following exposure to blood containing HBsAg the immunoglobulin can begiven as soon as possible after the exposure. For example, theimmunoglobulin preparation can be administered within 7 days followingthe exposure, for example, 24 hours following the exposure.

The immunoglobulin preparation of the invention may also be used in amethod for diagnosing a HBV-infected subject. This can be carried out byobtaining a body fluid sample, such as a blood sample, a lymph sample orany other body fluid sample, from said subject and contacting it withthe preparation of the invention under conditions which allow theformation of antibody-antigen complex as known in the art. Detection ofthe antibody-antigen complex level can be carried out by any methodsknown in the art. Negative control samples can also be tested. Eachsample which is significantly higher than the control sample indicateson an HBV infected subject.

The present invention also provides kits comprising containerscomprising the immunoglobulin preparation according to the invention.The kit can also contain instructions for use to the physician, healthcare professional, or the patient. The containers can be vials orpre-filled syringes which contain the preparation of the invention. Theimmunoglobulin can be provided in the kit as a solution or in a solidform, e.g. as lyophilized powder. The lyophilized powder can bereconstituted with injectable carries. The container can be of differentsizes and contain different volumes of the preparation, for example, thepreparation can have a volume of equal to or less than about 5 ml, forexample, about 3, 2, 1.5 or 1 ml. The kit may also contain a separatevial of the injectable carrier. In one embodiment of the invention, thereconstituted immunoglobulin comprises more than 635 IU/ml such as 700,1000, 1400, and 2000 IU/ml. Optionally, a syringe of an appropriate sizeis also included in the kit. Such a kit can contain the monthly dosagedivided into several vials containing the required dose, for example,the kit can contain 2 vials with a total dosage to be administered everysecond week.

The immunoglobulin preparations, methods and kits according to thepresent invention have at least one of the following advantages:facilitates ambulatory care such as home therapy (self administration)thereby improving the patient's life quality, has a high safety profilewith very few local or systemic adverse effects, attains an adequateconcentration of circulating antibodies more rapidly as compared toother known subcutaneous formulations, provides good protection ortreatment against HBV, enables fast administration, allows less frequentinjections per month as compared to other available formulations, can beadministered in a small volume dose, and reduces health care servicesand patients costs.

Yet another object of the invention is accomplished by providing amethod for treating, preventing and/or reducing hepatitis B viralinfection in a subject in need. The method comprises: a) providing ananti-hepatitis B immunoglobulin preparation, and b) administeringsubcutaneously to the subject the preparation of a) in a dose higherthan that applied by the intramuscular route to achieve anti-hepatitis Bantibodies in the circulation of the subject at a similar level andtiming as the intramuscular route. In one embodiment of the invention,the immunoglobulin preparation is as defined above.

In one embodiment of the invention, the subcutaneous dose is at least1.5-fold higher than the applied by intramuscular dose.

In another embodiment of the invention, the subject was previouslytreated by intravenous HBIG administration.

In another further embodiment of the invention, the anti-hepatitis Bantibodies level attained in the circulation of the subject is about orgreater than 100 mIU/ml, like for example 150, 200, 400 or 500 mIU/ml.In one embodiment of the invention, the anti-hepatitis B antibodieslevel attained in the circulation of the subject is higher than 100mIU/ml.

The proposed method can be carried out pre or post orthotopic livertransplantation, as prophylactic treatment to prevent viral hepatitis Brecurrence in a liver transplant recipient, in a subject born to ahepatitis B viral infected mother or in a subject who was exposed tohepatitis B virus.

The immunoglobulin preparation according to the present invention can beadministered in the dose and volumes as defined above.

In one embodiment of the invention, the therapeutic responses followingsubcutaneous administration according to the invention are comparable tothose induced by the intramuscular administration. The term “comparabletherapeutic response” means that the use of subcutaneous administrationaccording to the invention has substantially the same therapeuticresponse and/or prophylactic effect, as defined herein above, as theintramuscular administration.

It is another object of the present invention to provide apharmaceutical composition for subcutaneous treatment of OLT patientscomprising a pharmaceutically acceptable carrier and a dose ofanti-hepatitis B immunoglobulin which is higher than the dose in apharmaceutical composition formulated for intramuscular treatment of thesame indication. The term “indication”, as used herein, refers to amedical condition or symptoms associated with a medical condition, suchas, but not limited to, a disease, disorder or condition associated withhepatitis B virus, pre or post orthotopic liver transplantation,perinatal exposure of infants born to a hepatitis B viral infectedmother and/or following exposure to hepatitis B virus.

In one embodiment of the invention, the subcutaneous dose is at least1.5-folds higher, e.g. about 2 or 2.5-folds, than the intramusculardose.

In another aspect, the invention provides a method for treating,preventing and/or reducing a disease, disorder or condition associatedwith hepatitis B virus in a subject in need thereof, the methodcomprising a first dosing regimen of intramuscular administration ofanti-hepatitis B immunoglobulin, followed by a second dosing regimen,which comprises subcutaneously administering to the subject a dose ofanti-hepatitis B immunoglobulin that is higher than the dose employed bythe first dosing regimen of intramuscular administration, therebyobtaining anti-hepatitis B antibodies in the circulation of the subjectat a similar level and timing as in the first dosing regimen ofintramuscular administration.

In one embodiment of the invention, the timing is before the third,second or first day following subcutaneous administration.

In one embodiment of the invention, such transitioning can be carriedout in situations wherein intramuscular doses of HBIG arecontra-indicated.

In the methods of the invention, the immunoglobulin preparationaccording to the invention can be administered in combination with oneor more agents. Such agents may include, as non limiting example, antihepatitis B monoclonal antibodies, anti-viral agents, nucleosideanalogs, inhibitors of DNA polymerase, interferons such as interferonalpha-2a, adefovir dipivoxil, lamivudine (Epivir-HBV), adefovir(Hepsera), entecavir (Baraclude), telbivudine (Tyzeka), tenofovir(Viread) and the like.

In the case of such a combination therapy the antiviral agents can beprovided simultaneously with the immunoglobulin preparation orsequentially either before or after treatment with the immunoglobulinpreparation. In one embodiment of the invention, the antiviral agent andthe immunoglobulin preparation are formulated together and administeredsimultaneously. In another embodiment of the invention, the antiviralagent can be administered as a separate component either before or aftertreatment with the immunoglobulin preparation. The antiviral agent canbe administered in any suitable and/or conventional mode, including, butnot limited to, orally, topically, intravenously, intramuscularly andsubcutaneously. The antiviral agent can be used in a dosing which allowsoptimizing the efficacy of the treatment. In this regard, the desiredanti-hepatitis B antibodies in the circulation can be lower compared toa non combined treatment.

If desired, the immunoglobulin preparation of the invention can beadministered in combination with hepatitis B active vaccine such as witha live attenuated virus vaccine. Advantageously, such combinationtreatment may develop a greatest degree of protection against hepatitisB viral infection. The vaccine can be administered either before orafter treatment with the immunoglobulin preparation. The vaccine can beadministered in a different location than the administeredimmunoglobulin preparation. In one embodiment of the invention, thehepatitis B active vaccine is administered after treatment with theimmunoglobulin preparation. In one embodiment of the invention, thehepatitis B active vaccine is administered before treatment with theimmunoglobulin preparation.

In one embodiment of the invention any hepatitis B immunoglobulinpreparation is used and administered SC at a dose that is 1.5 foldhigher than that required by the intramuscular route. In one embodimentof the invention when converting the intramuscular dosing to thesubcutaneous dosing the dose is increased by 50%.

The disclosure of applications, patents and publications, cited above orbelow, is hereby incorporated by reference.

The following examples are illustrative but not limiting.

EXAMPLES Materials and Methods

Antibody analysis: anti-hepatitis B surface antigen (Anti-HBs)antibodies were analyzed by the automated immunoassay analyzer (AxSYMsystem; Abbott Diagnostics) using AUSAB kit [Microparticle EnzymeImmunoassay (META), Abbott Diagnostics (Abbott AxSYM® HBsAg Assay)].

Animal models: Two models were used as specified below. The studyprocedures were reviewed and approved by the Committee for EthicalConduct in the Care and Use of Laboratory Animals, at the HebrewUniversity, Jerusalem, Israel.

In both models the animals were housed for acclimation for at least 5days before surgical procedures. Animals were provided diet ad libitumand had free access to drinking water.

At the end of the study, surviving animals were euthanized by CO₂asphyxiation.

Rabbit model: Female NZW rabbits (Harlan Biotech, Israel, LTD), weighingabout 3 kg at study initiation were used. Four groups of rabbits wereused (4 rabbits per group). The constitution of the tested groups andthe pharmacokinetic parameters are listed in Table 1 below.

Test items: HepB IG 16% preparation containing anti-HBs antibodies at atiter of 1,400 IU/ml served as source material for all tested items.HepB IG 16% was diluted in 5% regular IVIG and 10% maltose to produce afinal 5% IgG preparation for IV administration. For SC/IM administrationthe source material was diluted in concentrated IVIG (20%) andglycine/saline diluent (2.25% glycine, 0.3% NaCl; at a pH of 6.5-7.0) toproduce a final 16% IgG preparation. Concentration and diafilterationwere carried out using Filtron, Minisette 30K.

The 5% and 20% IVIG preparations used to dilute the source materialcontained negligible titers of anti-HBs antibodies. The final dilutedtest items which were used for injection contained 109 and 565 IU/mlanti-hepatitis B antibodies for the IV and the SC/IM solutionsrespectively (see Table 1). The antibody titer was measured using theAbbott AxSYM® HBsAg assay according to manufacturer's instructions.

The test items were administrated at room temperature by one of thefollowing routes:

(i) Intravenous (IV) slow injection (about 1-2 ml/min; 1.8 ml/kg)through the ear vein followed by flushing with heparinised saline.

(ii) Subcutaneous (SC) injection (0.3 ml/kg) divided between two sites(back of the neck).

(iii) Intramuscular (IM) injection (0.3 ml/kg) divided between two sites(thighs).

Animals in the IV groups were tranquilized by xylazine 2% whileintroducing the test item.

Body weights were taken periodically and the animals were monitored forlocal adverse events at the injection sites.

TABLE 1 Constitution of test groups. Treatment Volume and Test ItemRoute Dosage IU/kg HepB IG IV 1.8 ml/kg, 196.2 5% 109 IU/ml HepB IG SC0.3 ml/kg, 169.5 16% IM 565 IU/ml Glycine/*NS SC 0.3 ml/kg — *NS—normalsaline.

Blood samples: Pre-treatment bleeding was obtained from the ear usingstandard techniques. The blood from the IV treated animals was takenfrom the contra-lateral ear that was not used for the IV administration.About 0.5 ml of blood was collected at each time point. The measurementswere carried out in the following time points 2, 6 and 8 hours and 1, 2,3, 4, 5, 6, 7 and 14 days following administration. Serum was separatedby centrifugation at 2500 g for 15 minutes at room temperature andtransferred into a new tube for storage at −18° C. or below untilassayed.

Cannulated rat model: Male HsdHan wistar rats (Harlan Biotech IsraelLtd) at the age of about 9-10 weeks and weighing 300 g at studyinitiation were used in this model (4 rats per group).

All surgical procedures were done under analgesia (Buprenorphine, S.C.,0.05 mg/kg) and anesthesia (induced by inhalation of 4% Isoflurane inoxygen and maintained with 1.0-2.0% Isoflurane).

In each of the animals external jugular vein was catheterized and thecatheter was secured in the vein with the aid of ligatures. The catheterwas then passed through a subcutaneous tunnel created by bluntdissection towards the skin on the dorsal aspect between the scapulasand secured by sutures or stainless steel wound clips.

Body weights were measured during acclimation, shortly before anesthesiainduction towards cannulation, prior to dosing and on 2, 4, 7 and 15days post-dosing.

Test items: 5% and 16% HepB IG preparations were prepared as indicatedabove. The final diluted test items used for injection contained 109 and282 IU/ml anti-hepatitis B antibodies for the IV and the SC/IM solutionsrespectively (see Table 2 below).

The test items were administered about twenty-four hours after Jugularcannulation. The constitution of the tested groups and thepharmacokinetic parameters are listed in Table 2. The test items wereadministrated at room temperature in one of the following routes:

(i) Intravenous (IV) bolus injection (1.8 ml/kg) through the cannula(jugular), followed by flushing with 0.2 ml of warmed saline.

(ii) Subcutaneous (SC) injection (0.6 ml/kg) over the left flank of theanimal.

(iii) Intramuscular (IM) injection (0.6 ml/kg) divided between two sitesto the right and the left thigh muscles of the animal.

TABLE 2 Constitution of test groups. Treatment Volume and Test ItemRoute Dosage IU/kg HepB IG IV 1.8 ml/kg, 196.2 5% 109 IU/ml HepB IG SC0.6 ml/kg, 169.2 16% IM 282 IU/ml

Blood samples: Pre-treatment bleeding point was obtained from theretro-orbital sinuses, under isoflurane anesthesia. After dosing, bloodsamples were obtained via the intravenous cannula, as long as thecannula was potent. About 0.25-0.3 ml of blood was collected at eachtime point. Following each blood collection from the cannula about 0.3ml of warmed physiological saline was injected. Alternatively, bloodsamples were obtained from the retro-orbital sinuses or by tailsectioning under general Isoflurane anesthesia. The measurements werecarried out in the following time points 1, 2, 4, 7 and 11 daysfollowing administration. At the final bleeding, day 15 post-dosing,blood was collected by cardiac puncture, under general anesthesia. Theblood sample was put into a tube containing 1 μl of Sodium Heparin (5000IU/ml). Each vial was gently mixed and plasma was separated bycentrifugation at 2500 g for 10 minutes at room temperature andtransferred into a new tube for storage at −18° C. or below untilassayed.

Example 1 Monitoring the Anti-Hepatitis B Antibody Levels in theCirculation Following Intravenous, Subcutaneous or IntramuscularAdministration of Hepatitis B Immunoglobulin

The present example was aimed to determine the level of circulatingantibodies following the different routes of administrations. Two animalmodels were used in these experiments: a rabbit model and cannulated ratmodel. In each model the animal was administered with the different testitems as indicated in Table 1 or 2 and blood samples were taken foranalysis of anti-hepatitis B antibody levels (as specified above). Eachdetermination is the average result of four animals.

FIG. 1 shows the recovery of anti-hepatitis B antibodies in the rabbit'sblood following administration of HBIG in the different routes ofadministrations. The level of antibodies on day one following IVadministration was assigned as a reference value (100%).

The results indicate that both IV and IM administration resulted in ahigh and rapid appearance of anti-hepatitis B antibodies in the animal'scirculation at day 1. IM administration achieved a maximum antibodylevel of about 70-80% of the IV level one day following administration.In contrast, SC administration resulted in a delayed appearance ofantibodies in the blood. About 35% of the IV level was attained one dayfollowing administration and a peak serum concentration of 53% wasobserved 3 days following administration.

FIG. 2 shows the level of the anti-hepatitis B antibodies in therabbit's blood during the first two weeks following administration ofthe HBIG preparation. It is apparent that the antibody levels in thecirculation in both IV and IM routes is substantially higher than thatobserved in the SC group on the first and second days followingadministration (2998 for IV and 2305 for IM compared with 1040 mIU/ml,for SC on day 1 following administration and 2032 for IV and 2016 for IMcompared with 1460 for SC mIU/ml, on day 2 following administration).

Similar results were found in the cannulated rat model. FIG. 3 shows therecovery of anti-hepatitis B antibodies in the rat's blood followingadministration of HBIG. The results are presented as percentage of thelevel of antibodies on day one following IV administration (100%).

As observed in the rabbit model, in the IV and IM route theanti-hepatitis B antibodies appear more rapidly than when givensubcutaneously. 34 and 38% of the IV level was attained on day 1 and 2following administration, respectively, for IM vs. 13 and 27% antibodyrecovery on day 1 and 2 following administration, respectively, for SCadministration (FIG. 3). SC injection exhibited a peak serumconcentration of about 30% about 4 days following administration.

While observing the antibody levels in the rat's circulation during thefirst two weeks following administration (FIG. 4) it is evident that SCadministration results in a lower antibody level in the blood on day 1and 2 following injection as compared to IM and IV administration (279compared with 2042 and 714 for SC, IV, and IM respectively, on day 1following administration and 549 compared with 1445 and 795 mIU/ml forSC, IV, and IM respectively, on day 2 following administration).

The results obtained herein collected during the observation period oftwo weeks show that the calculated average area under the curve (AUC) inboth models was similar in the SC and IM routes. These results are inline with previous reports (Thürmann et al. 2006 and Hooman at al.2008).

In addition, it is apparent that in both models intramuscularadministration resulted in about 2-fold increase in circulatinganti-hepatitis B antibodies one day following injection as compared tosubcutaneous administration (2305/1040 and 714/279 for the rabbit modelor cannulated rat model, respectively).

No adverse events occurred at the injection sites during the studyperiod in both animal models.

The above results indicate that as opposed to IV and IM, SCadministration is slowly released into the intravascular space with peakserum concentrations achieved 3 to 4 days after injection, i.e.administering an immunoglobulin composition in SC route results in adecreased intravenous bioavailability on the first two days followinginjection as compared to the two other routes of administration. Whencomparing the anti-hepatitis B antibodies in the circulation on day onefollowing administration, it is evident that IM administration lead toabout 2-fold increase in the antibody levels as compared to SCadministration. This indicates the advantage of administeringsubcutaneously a higher amount of antibodies compared to intramuscularinjection, since administering this amount will result in early adequateserum IgG levels.

1. A method for subcutaneous immunoglobulin hepatitis B therapy,comprising subcutaneously administering to a subject in need a dose ofanti-hepatitis B immunoglobulin that is higher than a dose ofanti-hepatitis B immunoglobulin administered by the intramuscular route,thereby achieving anti-hepatitis B antibodies in the circulation at asimilar level and timing as by the intramuscular route.
 2. The methodaccording to claim 1, wherein said subject is selected from the groupconsisting of an orthotopic liver transplantation (OLT) patient, anewborn to a viral infected mother, and a subject exposed to the virus.3. The method according to claim 1, wherein said subcutaneous dose is atleast 2-fold higher than the dose administered by the intramuscularroute.
 4. The method according to claim 1, wherein said subcutaneousdose is higher than 2170 IU.
 5. The method according to claim 1, whereinsaid subcutaneous dose is equal to or higher than 3000 IU.
 6. The methodaccording to claim 1, wherein said subcutaneous administering is carriedout by injection.
 7. The method according to claim 1, wherein saidsubcutaneous administering is carried out by a single dispensation. 8.The method according to claim 1, wherein said subcutaneous dose has atiter of anti-hepatitis B antibodies that is greater than 635 IU/ml. 9.A method for treating, preventing and/or reducing a disease, disorder orcondition associated with hepatitis B virus, comprising administering toa subject in need thereof a dose of anti-hepatitis B immunoglobulinsufficient to obtain a predetermined level of anti-hepatitis Bantibodies in the circulation of said subject, the improvement whereinthe anti-hepatitis B immunoglobulin is administered by the subcutaneousroute in a dose that is at least 1.5 times higher than the intramuscularformulated dose needed to obtain said predetermined level ofanti-hepatitis B antibodies in the circulation of said subject, whereinsaid predetermined level is obtained in the circulation by thesubcutaneous dose at a similar timing as in said intramuscularformulated dose.
 10. The method according to claim 9, wherein saidpredetermined level of anti-hepatitis B antibodies in the circulation isgreater than 100 mIU/ml.
 11. The method according to claim 9, whereinsaid timing is before the third, second or first day followingsubcutaneous administration.
 12. The method according to claim 9,wherein said subcutaneous administration is carried out by injection.13. The method according to claim 9, wherein said subcutaneousadministration is carried out by a single dispensation.
 14. The methodaccording to claim 9, wherein said subject was previously treated by adosing regimen of intravenous administration of anti-hepatitis Bimmunoglobulin.
 15. The method according to claim 9, wherein saidsubcutaneous dose has a titer of anti-hepatitis B antibodies that isgreater than 635 IU/ml.
 16. A method for preventing hepatitis B viralinfection recurrence in a subject who underwent orthotopic livertransplantation (OLT), comprising administering to said subject a doseof anti-hepatitis B immunoglobulin sufficient to obtain a predeterminedlevel of anti-hepatitis B antibodies in the circulation of said subject,the improvement wherein the anti-hepatitis B immunoglobulin isadministered by the subcutaneous route in a dose that is at least 1.5times higher than the intramuscular formulated dose needed to obtain asimilar level of anti-hepatitis B antibodies in the circulation of saidsubject, wherein said predetermined level is obtained in the circulationby the subcutaneous dose at a similar timing as in said intramuscularformulated dose.
 17. The method according to claim 16, wherein saidsubcutaneous administering is carried out by injection.
 18. The methodaccording to claim 16, wherein said subcutaneous administering iscarried out by a single dispensation.
 19. A method for treating,preventing and/or reducing a disease, disorder or condition associatedwith hepatitis B virus in a subject in need thereof, the methodcomprising a first dosing regimen of intramuscular administration ofanti-hepatitis B immunoglobulin, followed by a second dosing regimen,which comprises subcutaneously administering to the subject a dose ofanti-hepatitis B immunoglobulin that is higher than the dose employed bythe first dosing regimen of intramuscular administration, therebyobtaining anti-hepatitis B antibodies in the circulation of said subjectat a similar level and timing as in the first dosing regimen ofintramuscular administration.
 20. The method according to claim 19,wherein said timing is before the third, second or first day followingsubcutaneous administration.